Role of Stress-Response Protein gp96 in the Persistence of Rheumatoid Arthritis

描述（由申请人提供）：先天和适应性免疫系统在类风湿关节炎（RA）的发病机制中很重要。Toll样受体（TLRs）对适应性免疫的产生很重要，包括T细胞和B细胞的激活。然而，tlr在RA的持续和破坏中的作用尚不清楚。我们最近证明，表达突变Fas受体（lpr）的小鼠，发生较少的频率和较轻的胶原诱导关节炎（CIA）。我们发现了一种新的机制，即Fas-FasL与巨噬细胞（M&s）相互作用，虽然不诱导凋亡，但通过TLR4途径增强了活化。我们发现完整的Fas- FasL信号通路促进了CIA的诱导，并增强了TLR4连接诱导的NF-:B活化和细胞因子的表达。M&s和滑膜成纤维细胞在RA的发病机制中起关键作用。RA关节中的M&s同时表达Fas和FasL，并与其他M&s和滑膜成纤维细胞密切接触。这些观察结果表明，在RA关节中，Fas-FasL相互作用通过降低激活阈值，可能使滑膜M&s对TLR配体的激活敏感。我们的初步研究还表明，与对照M&s相比，从RA关节分离的M&s对微生物TLR2和TLR4配体的反应性增强，这表明TLR信号可能在RA中很重要。我们的初步数据还表明，96- kda糖蛋白96 （gp96）是一种内质网应激反应蛋白，在RA关节中高表达，并通过TLR2激活M&s，非凋亡性Fas-FasL相互作用促进gp96激活M&s。我们还证明了另一种潜在的TLR配体HSP22，通过TLR4激活，在RA滑液中含量丰富。综上所述，这些观察结果表明，mas之间以及mas与滑膜成纤维细胞之间的Fas-FasL相互作用可能使关节内的局部细胞对内源性TLR配体（如gp96或HSP22）的激活敏感，从而导致自我延续的慢性炎症。这项应用的最重要的假设是内源性TLR配体的局部释放促进关节炎从自限性疾病发展为慢性疾病，并且Fas-FasL相互作用增强了这种进展。具体而言，我们假设：1)使用gp96和HSP22，通过TLR结扎的RA滑膜M&s的激活将大于正常M&s或来自银屑病关节炎等其他形式炎性关节炎（OIA）患者滑液的M&s，而RA滑膜成纤维细胞的激活将大于骨关节炎（OA）患者的滑膜成纤维细胞；2)内源性TLR配体介导的最大TLR2或tlr4激活需要Fas-FasL相互作用；3) gp96（或HSP22）的表达将自限性关节炎转化为慢性进行性疾病，依赖于TLRs，在存在而不是不存在完整的Fas信号的情况下；4)gp96（和/或HSP22）的中和将改善CIA。

DESCRIPTION (provided by applicant): The innate and adaptive immune systems are important in the pathogenesis of rheumatoid arthritis (RA). Toll- like receptors (TLRs) are important for the generation of adaptive immunity, including T and B cell activation. However the role of TLRs in the persistence and destruction of RA is not known. We recently demonstrated that mice expressing a mutant Fas receptor (lpr), developed less frequent and less severe collagen induced arthritis (CIA). We identified a novel mechanism whereby Fas-FasL interactions on macrophages (M&s), while not inducing apoptosis, enhanced activation through the TLR4 pathway. We demonstrated that intact Fas- FasL signaling promoted the induction of CIA and enhanced the NF-:B activation and cytokine expression induced by TLR4 ligation. M&s and synovial fibroblasts are critical in the pathogenesis of RA. M&s in the RA joint, express both Fas and FasL and are in intimate contact with other M&s and with synovial fibroblasts. These observations suggest that, in the RA joint, Fas-FasL interactions, by lowering the threshold for activation, may sensitize synovial M&s to activation by TLR ligands. Our preliminary studies also demonstrate that M&s isolated from the RA joint demonstrate increased responsiveness to microbial TLR2 and TLR4 ligands compared to control M&s, suggesting that TLR signaling might be important in RA. Our preliminary data also demonstrate that the 96-kDa glycoprotein 96 (gp96), an endoplasmic reticulum stress response protein, is highly expressed in the RA joint, and activates M&s through TLR2 and that non-apoptotic Fas-FasL interactions promote M& activation by gp96. We also demonstrate that another potential TLR ligand, HSP22, which activates through TLR4, is abundant in RA synovial fluids. Together these observations suggest that Fas-FasL interactions between M&s, and perhaps between M&s and synovial fibroblasts, may sensitize cells locally within the joint to activation by endogenous TLR ligands, such gp96 or HSP22, resulting in a self- perpetuating chronic inflammation. The overriding hypothesis of this application is that the local release of endogenous TLR ligands promotes the progression of arthritis from a self-limited to a chronic disease, and that this progression is enhanced by Fas-FasL interactions. Specifically, we hypothesize that: 1) Employing gp96 and HSP22, the activation of RA synovial M&s through TLR ligation will be greater than observed with normal M&s or those from the synovial fluid of patients with other forms of inflammatory arthritis (OIA) such as psoriatic arthritis, while the activation of RA synovial fibroblasts will be greater than observed with synovial fibroblasts from patients with osteoarthritis (OA); 2) that Fas-FasL interactions are required for maximal TLR2 or TLR4-mediated activation by endogenous TLR ligands; 3) that the expression of gp96 (or HSP22) converts self-limited arthritis to chronic progressive disease, dependent upon TLRs, in the presence, but not the absence, of intact Fas signaling and that 4) that neutralization of gp96 (and/or HSP22) will ameliorate CIA. PUBLIC HEALTH RELEVANCE. Rheumatoid Arthritis affects at least 1% of the population and there are many patients for whom therapy is inadequate. The work proposed in this study is aimed at identifying novel mechanisms contributing to the pathogenesis of rheumatoid arthritis, which would define new therapeutic targets.